cows in semi-arid conditions
PAPER SUBMITTED TO JOURNAL OF DAIRY SCIENCE (ATTACHMENT C)
INJECTABLE TRACE MINERALS IN DAIRY COWS
Effect of injectable trace mineral supplementation on peripheral polymorphonuclear leukocyte function, oxidative stress, health, and performance of dairy cows in semi-arid conditions
Silva T. H.1, 2, I. Guimaraes1, P. R. Menta1, L. Fernandes1, D. Paiva1, T. L. Ribeiro1, M. L.
Celestino1, A. Saran Netto2, M. A. Ballou1, V. S. Machado1*
1Department of Veterinary Sciences, Texas Tech University, Lubbock, TX 79415
2Department of Animal Science, School of Animal Science and Food Engineering, University of Sao Paulo, Pirassununga, SP 13635-900 Brazil
*Corresponding author: Vinicius Silva Machado, Department of Veterinary Sciences, College of Agricultural Sciences and Natural Resources, Texas Tech University, Lubbock, TX 79409, United States., Phone: 806 834-4695, Fax: 806 742-2836, e-mail: [email protected]
ABSTRACT
The objective of this study was to evaluate the effect of 3 subcutaneous injections of 15 mg/mL of Cu, 5 mg/mL of Se, 60 mg/mL of Zn, and 10 mg/mL of Mn at dry-off, 20 days prior to expected day of calving, and 35 days postpartum, on immunity, oxidative stress, health and performance of dairy cows undergoing the transition period in high temperature humidity index (THI). The study was conducted on 2 commercial Holstein dairy farms located in west Texas. A total of 923 multiparous cows were randomly allocated into 1 of 2 treatment groups: injectable trace mineral supplement (ITMS) or control. Blood samples were collected at enrollment, and at 3
± 1, 7 ± 1, 10 ± 1, and 35 ± 3 DIM to evaluate hematology, polymorphonuclear leukocyte (PMNL) function, circulating biomarkers of oxidative stress, antioxidant enzymes, and circulating haptoglobin (Hp). Trace mineral supplemented cows had a lower incidence of metritis compared to control group (ITMS = 8.6% vs. control = 12.6%). Additionally, ITMS-treated cows had lower incidence of stillbirth compared to control cows (ITMS = 1.5% vs. control = 3.7%). However, ITMS did not influence milk yield, reproductive performance, and culling. Blood analysis revealed that ITMS did not influence the white blood, neutrophil, and lymphocyte counts, but neutrophil to lymphocyte ratio was greater for ITMS-treated cows in comparison to control cows. Additionally, ITMS cows had improved PMNL function during the post-partum period due to increased proportion of PMNL that performed phagocytosis and oxidative burst and increased the intensity of oxidative burst. Conversely, control cows had increased L-Selectin expression on PMNL surface. Concentration of serum antioxidant enzymes and oxidative stress biomarkers were not affected by ITMS treatment, but ITMS treatment tended to increase Hp concentration at 10 d after calving. In conclusion, ITMS improved PMNL function and reduced incidence of metritis and stillbirth parturition in dairy cows undergoing the transition period in high THI conditions.
However, these findings did not translate into improved milk yield, reproductive performance, and survivability.
Key Words: trace minerals, dairy cows, immune function, health
INTRODUCTION
During the periparturient period, dairy cows experience a substantial increase in oxygen consumption that results in elevated production of reactive oxygen species (ROS; Sordillo, 2016).
Therefore, an imbalance between the production of ROS and the availability of antioxidants defenses may expose these animals to oxidative stress (SORDILLO; AITKEN, 2009).
Additionally, heat stress has been reported to aggravate this scenario, whereas cows can experience higher degrees of feed intake depression (ADIN et al., 2009), increased degree of oxidative stress (BERNABUCCI et al., 2002; SAFA et al., 2019), and immunosuppression (LACETERA et al., 2005; LECCHI et al., 2016; SAFA et al., 2019). Therefore, high proportion of puerperal disorders have been observed in dairy cows undergoing the transition period in heat stress conditions (GERNARD et al., 2019). Heat stress in dairy cows is particularly important dairy herds located in semi-arid regions. For instance, in the Southwest region of the High Plains in the United States or Northern Mexico, where the dairy industry is an important part of the local economy, the average Temperature Humidity Index (THI) during the summer is above 68, commonly used as the threshold for stress in dairy cows (ZIMBELMAN et al., 2009).
Providing shade and soaking lines are strategies commonly adopted to improve the performance and immune status of postpartum dairy cows undergoing heat-stress (DO AMARAL et al., 2011). Also, supplementation of trace minerals has been studied as alternative strategies to alleviate heat stress' detrimental effects on immunity, antioxidant status, and performance of chicken (ABD EL-HACK et al., 2017; RAJKUMAR et al., 2017), and sheep (ALHIDARY et al., 2015). Hence, it is plausible that trace mineral supplementation can also help to mitigate the burden of heat stress in dairy cows. The use of injectable trace mineral supplementation (ITMS) with Zn, Mn, Cu, and Se improved health (MACHADO et al., 2013) and antioxidant status (MACHADO et al., 2014) of dairy cows supplemented at dry-off, 30 days prior to calving, and during the second month of lactation. However, postpartum polymorphonuclear leukocyte (PMNL) function, milk yield, and reproductive performance were not influenced by ITMS. Those studies were performed in dairy herds located in NY, during cold weather conditions. Hence, the use of ITMS in cows in semi-arid conditions, with added stressful conditions such as increased THI during summer months, could lead to different outcomes. For example, during a stressful even (aflatoxin challenge) ITMS decreased inflammation and oxidative stress in dairy cows (PATE; CARDOSO, 2018).
Therefore, the objective of this study was to evaluate the effect of an injectable trace mineral supplementation containing copper, selenium, zinc, and manganese at dry-off, 20 days prior to expected day of calving, and 35 days postpartum on incidence of postpartum diseases, milk yield, reproductive performance, culling, hematological parameters, PMNL activity, concentrations of oxidative stress markers, and circulating haptoglobin in dairy cows undergoing the transition period in high THI.
MATERIALS AND METHODS
Farms, management, and environmental data
All experimental procedures were approved by the Texas Tech University Institutional Animal Care and Use Committee (#19045-05). This study was conducted on 2 commercial Holstein dairy farms (Farm A and Farm B), located in west Texas. Farms were selected based on their geographical location (near Lubbock, TX), and willingness to participate in the study. Cows were enrolled from 30 April until 23 July 2019; the follow-up period continued until 13 November 2019. The enrollment period was selected to maximize the number of days with THI > 68 during the transition period. The environmental information was assessed from the Lubbock Airport’s meteorological station because its short geographic distance from both dairies (67 and 48 kilometers from farms A and B, respectively). Daily temperature and relative humidity data were downloaded from April 30th through November 13th, 2019. The THI throughout the study period was calculated from the equation developed by the National Research Council (NRC, 1971) below as indicated by Dikmen & Hansen (2009):
THI = (1.8 × T°C + 32) – [(0.55 − 0.0055 × RH%) × (1.8 × T°C − 26)],
where T = ambient dry bulb temperature in °C, and RH% = relative humidity. Meteorological data are presented in Figure 1.
Farm A milked 2,700 Holstein cows 2 times per day in a double 30-stall parallel milking parlor. The cows were housed in dry-lot pens, consisting of non-vegetated open lots (i.e., corrals) with shaded areas. Farm B milked 3,700 Holstein cows three times daily in a 70-stall rotary milking parlor. During the dry period, the animals were house in dry-lot pens, consisting of non-vegetated open lots. After calving, the cows were housed in free-stall barns, with concrete stalls and bedded with manure solids. At both farms, the animals were fed a total mixed ration (TMR) ad libitum with free access to water. According to herd characteristics, the diets were formulated to meet or exceed the NRC (2001). Composite total mixed ration samples from pre-fresh and lactation diets were submitted to a commercial laboratory (Cumberland Valley Analytical Services, Inc.). Dry matter, crude protein, acid detergent fiber, neutral detergent fiber, and macro and micro minerals for analyzed by wet chemistry methodology. Nutrient contents of the diets are described in Table 1.
Sample size calculation
Based on previously reported data (Machado et al., 2013), we assumed that ITMS would decrease the incidence of clinical mastitis from 26% to 18%. Considering a significance level of α=0.05 and a power of 80%, at least 420 cows would have to be enrolled in each treatment group.
Accounting for 10% attrition, 923 animals were enrolled in the study (n = 449 and n = 474 for control and ITMS group, respectively). With this sample size, we were able to detect a milk production difference between the groups of 1.1 kg/d with a S.D. of 6 kg/d.
Inclusion criteria, treatment allocation, case definitions, and data collection
Dry cows were included in the study based on expected calving date (between July and September). A completely randomized clinical study blocked by farms was performed. A total of 923 multiparous cows (from 2 dairies) were randomly allocated into 1 of 2 treatment groups:
injectable trace mineral supplement (ITMS) or control. Randomization was completed in Excel (Microsoft) and imported into the farm’s management software program. Cows that were randomly assigned to the ITMS group received 3 subcutaneous injections of 15 mg/mL of Cu, 5 mg/mL of Se, 60 mg/mL of Zn, and 10 mg/mL of Mn (Multimin 90, Multimin North America, Fort Collins, CO). A blanket dose (7 ml) was administered for all the animals enrolled in ITMS group. We followed the manufacturer’s recommendation, administrating for cattle over 2 years of age, at dry-off (208 ± 3 days of gestation), 260 ± 3 d of gestation, and at 35 ± 3 days postpartum. Pregnant heifers were not enrolled in the study because ITMS was not beneficial to cows entering the lactating herd (Machado et a., 2013).
Postpartum diseases were diagnosed and treated by trained farm personnel, who were blinded to treatments. Metritis was defined as the presence of fetid, watery, red-brown uterine discharge (SHELDON et al., 2006). Clinical mastitis was defined by the diagnosis of abnormal changes in the udder and/or milk (LAGO et al., 2011). Retained fetal membranes was defined as the failure to release fetal membranes within 24 h of calving (KELTON; LISSEMORE; MARTIN, 1998). Stillbirth was defined as the death of a calf occurring just before, during, or within 48 h of parturition (PHILIPSSON et al., 1979). Data regarding health traits, reproduction outcomes, milk yield, and survivability were extracted from Bovisynch (Dairy LLC, WI) and DairyComp 305 (Valley Agricultural Software, Tulare, CA) database on farms A and B, respectively.
Blood collection and analysis
For a subset of 134 cows from Farm A, blood samples were collected for hematology purposes, PMNL function, circulating biomarkers of oxidative stress, antioxidant enzymes, and circulating haptoglobin (Hp). Blood samples were collected at enrollment, and at 3 ± 1, 7 ± 1, 10
± 1, and 35 ± 3 DIM by puncture of the coccygeal vessels using a vacutainer tube without anticoagulant, a vacutainer tube with lithium heparin, a vacutainer tube with potassium EDTA, and a 20-gauge x 2.54-cm vacutainer needle (Becton, Dickinson and Company, Franklin Lakes, NJ). After collection, the heparinized blood was stored in an ice chest with no ice at ambient temperature, to preserve their phagocytic and oxidative burst capacity (SELLERS; HULBERT;
BALLOU, 2013). The EDTA and coagulated blood samples were immediately placed on ice.
Samples were analyzed or processed within 3 h after collection. The blood samples without anticoagulants were centrifuged at 2000 x g for 15 min at 4 °C for serum separation, and frozen at
−80 °C. The heparinized and EDTA blood samples were processed in the same day of collection for measures of hematology and ex vivo PMN responses.
Leukocyte count and differentials were performed using a hematology analyzer (IDEXX Procyte DX, Westbrook, ME), and the variables of interest were white blood cells, monocytes, neutrophils, lymphocytes, and neutrophil to lymphocyte ratio. Flow cytometry was used to determine the phagocytosis and oxidative burst capacity of PMNs and the quantification of their adhesion molecule L-selectin (CD62 L) as previously described with minor protocol modifications (HULBERT et al., 2011). To measure the phagocytic and oxidative burst capacity, 100 μL of heparinized whole blood were incubated for 15 min in an ice bath. After this initial incubation, 20 μL of a 100 μM solution of dihydrorhodamine (Invitrogen, Carlsbad, CA), and 20 μL of a 109 cfu/mL propidium iodide labeled E. coli suspension were added to the blood and then incubated in a 38.5 °C water bath for 10 min (negative controls were incubated in an ice bath for 10 min).
Then, samples were immediately placed on an ice bath for 5 min, and erythrocytes were hypotonically lysed, and washed with PBS. Dual-color flow cytometry was performed using an Attune flow cytometer (Life Technologies, Thermo Fisher Scientific Inc., Waltham, MA). The PMN population was gated using forward and side scatter plots. The mean fluorescence intensity (MFI) and proportion of PMNs that performed phagocytosis and oxidative burst were acquired using the optical filters BL3 (excited by a 488 nm laser on a 695/40 filter) and BL1 (excited by a 488 nm laser on a 530/30 filter), respectively. Negative controls were used to determine negative
and positive signals on the BL1 by BL3 scatterplot used to assess PMNs that performed phagocytosis and oxidative burst. To determine the expression of the adhesion molecule L-selectin, 50 μL of EDTA blood samples were mixed with 50 μL of PBS containing 1 μg of a monoclonal antibody mouse IgG1-isotype (catalog number: BOV2046, clone: BAQ92A;
Veterinary Microbiology and Pathology Monoclonal Antibody Center, Pullman, WA). After a 1 h incubation in an ice bath, erythrocytes were hypotonically lysed. After centrifugation, the leukocyte pellet was resuspended in a 50 μL solution of fluorescein-labeled secondary antibody at a 1:400 dilution (F(ab′)2 anti-mouse IgG:FITC; AbD Serotec Raleigh, NC) and incubated for 1 h in an ice bath. After a PBS wash, samples were analyzed using single-color flow cytometry. The PMN population was gated as previously described, and the MFI for L-selectin was gathered using BL-1. Data were analyzed using Attune Cytometric software (Life Technologies, Thermo Fisher Scientific Inc.).
Commercial kits (Cayman Chemical, Ann Arbor, MI) were used to assess the following markers of oxidative stress: Glutathione Peroxidase (#703102), Superoxide Dismutase (#706002), and Thiobarbituric Acid Reactive Substances (#10009055). Serum Hp levels concentration was determined using a colorimetric assay via quantification of the haptoglobin/hemoglobin complex by the estimation of differences in peroxidase activity (MAKIMURA; SUZUKI, 1982). Assays were performed in 16 × 100 borosilicate tubes. Briefly, 5 μL of serum sample or deionized water (blank) were added to 7.5 mL of a solution containing 0.6 g/L of O dianisidine, 13.8 g/L of sodium phosphate monobasic, and 0.5 g/L EDTA (pH = 4.1). Immediately, 25 μL of 0.3 g/L bovine hemoglobin solution was added to each assay, followed by a water bath incubation at 37 °C for 45 min. After incubation, 100 μL of freshly prepared 156 mM hydrogen peroxidase solution was added to each assay. Samples were incubated at room temperature for 60 min. Then, 200 μL of each assay was transferred to a 96-well polystyrene flat-bottom microplate. Optical density at 450 nm was measured on the Epoch2 Microplate Spectrophotometer (BioTek). Finally, the final OD of each assay was subtracted by the blank assay OD. Optical density data was converted to a concentration unit (μg/mL) using standard curves generated by serial dilutions of a sample of known concentration determined by a commercially available ELISA kit following the manufacturer’s instructions (Life Technologies) as previously described (COOKE;
ARTHINGTON, 2013). The intra and inter-assay coefficient of variation for serum Hp were 1.8
% and 5.9 %, respectively.
Statistical analysis
Descriptive statistics analysis was undertaken in SAS using the FREQ procedure of SAS (v.9.4, SAS Institute Inc., Cary, NC). The odds of clinical metritis, clinical mastitis, retained placenta, stillbirth, and pregnancy per AI (P/AI) at first service was assessed by fitting the data using the GLIMMIX procedure of SAS (v.9.4, SAS Institute Inc., Cary, NC). Variables offered to the models included treatment (which was forced into all the models), parity (2, >2), twins, and previous days carried calf. Additionally, the variable farm was included in all models as a random effect. The independent variables and their respective interactions were kept when P < 0.10.
The effect of treatment on reproduction and survival was analyzed by Cox’s Proportional Hazard using the proportional hazard regression procedure in SAS (v.9.4, SAS Institute Inc., Cary, NC). For analysis of reproduction, cows were right-censored if not diagnosed pregnant before culling, death, or the end of the data-collection period. Variables offered to the models included treatment, parity (2, >2), twins, and previous days carried calf. The independent variables and their respective interactions were kept when P < 0.10.
The effect of ITMS on monthly milk yield during the first 180 DIM was evaluated through a repeated measures model fitted by multiple mixed linear models using the MIXED procedure of SAS (v.9.4, SAS Institute Inc., Cary, NC). The model included the fixed effects of treatment, time, parity (2,>2), twins, previous days carried calf. The independent variables and their respective interactions were kept when P < 0.10. The farm variable was included as a random effect. To account appropriately for within-cow correlation, the error term was modeled by a compound symmetry (CS) covariance structure due to its smallest Bayesian Information Criterion (BIC) value.
To evaluate the effect of ITMS on cell blood count, PMN phagocytosis and oxidative burst activity, SOD, GPx, TBARS, and Hp throughout the five blood collection days (-72, 3, 7, 10, and 35 days relative to calving), repeat measures models, ordered by time, were fitted by multiple mixed linear models using the MIXED procedure of SAS (v.9.4, SAS Institute Inc., Cary, NC).
The experimental unit was the cow. The normality of the residuals was analyzed with normal probability and box plots visualization. To account appropriately for within-cow residues correlation, a spatial (power) covariance structure (SP[POW]) was applied for all models. This variance-covariance structure is indicated for unequally spaced data collection and assumes
correlations decline as a function of time. For multivariate models above, independent variables and their respective interactions were kept when P < 0.10. The variable treatment, time, and their interaction were forced into all statistical models even in the absence of statistical significance.
Days of gestation at enrollment, parity (2,>2), and the own variable value from -72 days relative to calving, were offered to all models as a covariate to control confounding effects. For all the analyses, differences detected at P ≤ 0.05 were considered significant, and differences at 0.05 < P
< 0.10 were considered a tendency toward statistical significance.
RESULTS
Descriptive statistics
A total of 956 animals were enrolled in the study. From these, 33 cows were excluded due to loss to follow-up, with 24 cows being from Farm A (control group = 11 and ITMS = 13) and 9 cows being from Farm B (control group = 2 and ITMS group = 7). A total of 923 cows from initial 956 remained in the study. Descriptive statistics regarding the number of animals enrolled in parity 2 and >2 and average gestation length (days) at enrollment are presented in Table 2.
Effect of ITMS on health, reproductive performance, milk yield, and culling
The effect of treatment on health and P/AI at first service is presented in Table 3. Briefly, supplementing cows with injectable trace minerals did not improve the odds of mastitis (P = 0.38) and retained placenta (P = 0.66) compared with non-supplemented cows. However, trace mineral supplemented cows had a lower incidence of metritis compared to control group (OR = 0.68, 95%
CI = 0.4 – 1.0, P = 0.05). Additionally, ITMS-treated cows had lower incidence of stillbirth compared to control cows (OR = 0.41, 95% CI = 0.17-1.01, P = 0.05).
Injectable trace mineral supplementation did not influence milk yield, reproductive performance, and culling. The average milk yield during the first 6 months of lactation for cows enrolled in control and ITMS group was 43.9 kg/d and 43.4 kg/d, respectively (P = 0.17), and there was no interaction between treatment and month of lactation (P = 0.92). Additionally, no effect of ITMS was detected during the follow-up period. The treatment did not improve P/AI at first service (OR = 1.03, 95% CI = 0.76 – 1.39, P = 0.87) or hazard of pregnancy up to 150 DIM compared to control cows (HR = 0.99, 95% CI = 0.88 – 1.17, P = 0.89). Regarding survivability, the hazard of culling up to 300 DIM was similar between the groups (HR = 1.23, 95% CI = 0.93 – 1.62, P = 0.14).
Effect of ITMS on hematology, PMNL function, oxidative stress biomarkers and haptoglobin The effect of ITMS on white blood cells, monocyte, neutrophil, and lymphocyte counts, neutrophil to lymphocytes ratio are presented in Table 4. The ITMS treatment did not influence the white blood, neutrophil, and lymphocyte counts during the study. However, neutrophil to
lymphocyte ratio was greater for ITMS-treated cows in comparison to control cows throughout the study (P = 0.03).
The ITMS treatment improved the innate immunity during the post-partum period due to increased proportion of PMNL that performed phagocytosis (P < 0.01) and oxidative burst (P <
0.01) and increased the intensity of oxidative burst (P = 0.05; Figure 2A, B, D). Conversely, the expression of the L-Selectin on PMNL surface was increased in control cows (P = 0.04; Figure 2E).
Although we observed treatment differences in some health outcomes, ITMS did not influence serum antioxidant enzymes concentration and oxidative stress biomarkers concentration after calving (P > 0.05; Table 4). However, control cows tended to present higher serum haptoglobin concentration at 10 d after calving (P = 0.09; Figure 2F).
DISCUSSION
The objectives of this study were to evaluate the effect of ITMS on the health, performance, and biomarkers of postpartum immune and antioxidative status in dairy cows undergoing their transition period with THI > 68. The meteorological analysis presented in Figure 1 demonstrates
The objectives of this study were to evaluate the effect of ITMS on the health, performance, and biomarkers of postpartum immune and antioxidative status in dairy cows undergoing their transition period with THI > 68. The meteorological analysis presented in Figure 1 demonstrates